Olivia Gavard

Modeling and analysis of the interactome of human Aurora A kinase

Abstract:
The kinase Aurora A is an essential mitotic cell cycle protein. Aurora A is necessary for mitotic entry and for the maturation and separation of centrosomes. It participates in mitotic spindle assembly and chromosome biorientation, and it is essential for the completion of cytokinesis. Furthermore, Aurora A activity is necessary for the equal distribution of mitochondria to daughter cells and, through its role in the alternative splicing of mRNA of apoptotic factors, it provides a link between cell cycle control and apoptosis. Beyond its mitotic functions, several recent studies suggest that Aurora A is also important during interphase. Notably, it influences microtubule dynamics, promotes cell migration and polarity control and is essential for primary cilia disassembly. Reflecting the fact that Aurora A is found to be up-regulated in many cancers, deregulation of Aurora A activity can result in an aberrant cell cycle, ultimately leading to malignant transformation of cells. The crucial regulation of Aurora A’s numerous functions is achieved through its interaction with several protein partners, which modulate its activity, localisation and stability. Aurora A in turn phosporylates a number of them, thus regulating their activity, localisation and stability. However, the known interactions of Aurora A cannot explain all the phenotypes that have been described of its deregulation.To better understand the functions of Aurora A, the regulation mechanisms governing it, and to expose its multiple roles in the cell, I have built and analysed an Aurora A interactome using tandem affinity purification coupled with mass spectrometry. This resulted in the identification of 477 potential interacting partners, of which, 180 were determined to have a high probability of interacting directly with the kinase.In-depth bioinformatic analysis of this interactome has revealed the associated partners to be related to mitochondria and mRNA splicing, highlighting the potential involvement of Aurora A in these mechanisms. To validate the interactome, two of the proteins identified in this study, WDR62 and CEP97, were examined in detail. Here I show that these two proteins colocalise with Aurora A, and are phosphorylated by the kinase.WDR62 is implicated in microcephaly and is deregulated in certain cancers. I have shown that Aurora A phosphorylates WDR62 during mitosis, and that this phosphorylation is necessary for its localisation to the centrosomes. CEP97 is a poorly charactarised protein of the primary cilium, abnormalities of which are associated with ciliopathies. I have shown that Aurora A phosphorylates CEP97 in vitro, and that the inhibition of Aurora A activity in vivo perturbs the localisation of CEP97 to cilia and centrosomes.This study has identified a number of new Aurora A-interacting proteins, implicating the kinase with novel functions. These functions, related to mitochondria and mRNA splicing have opened up a new area for further investigation.

PhD in co-tutelle :
Université de Rennes1 (France) et Université Laval de Quebec (Canada)

Financial support :
French Fondation for Medical Research (FRM) & The Quebec Network for Research on Protein Function, Engineering, and Applications (PROTEO)

Committee :  : December 19th 2015
Florence GONNET
Patrick MERALDI
Bernard DUCOMMUN
Arnaud DROIT (PhD co-director)
Guy POIRIER (PhD co-director)
Claude PRIGENT (PhD co-director)

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